1. Field of the Invention
The present invention relates to conjugates of a protein having at least one stereoprotected mercapto group, and a bifunctional ligand capable of coupling to the mercapto group and of chelating with a metal. Specifically, such conjugates are useful for labeling proteins with metals, and can consequently be utilized for the detection and/or therapy of disease states.
2. Description of the Prior Art
Interest in the art of metal chelates and in methods for forming metal chelate-protein conjugates for diagnostic and therapeutic purposes continues. Representative type chelates and conjugates and methods for forming conjugates are disclosed, inter alia, in U.S. Pat. Nos. 4,454,106; 4,472,509; 4,339,426; 4,824,986; 4,831,175; 5,124,471; in EPA 0 279 307 and in German patent 1.155,122. Other proteins including antibodies, monoclonal antibodies and fragments thereof, monoclonal antibodies and fragments thereof which have been structurally altered by recombinant DNA techniques (i.e., chimeric antibodies), polyclonal antibodies, antigens, blood proteins, or proteins bound to blood lymphocytes or other cells can also be employed in the formation of conjugates.
A method for synthesis of bifunctional metal chelates for conjugation to proteins involves reduction of amino acid amides to ethylenediamines to form monosubstituted derivatives which are converted to bifunctional ethylenediaminetetraacetic acid (EDTA) chelates by alkylation with haloacetic acid. (Yeh et al., Anal. Biochem. 100:152 (1979)). Similarly, a monosubstituted diethylenetriamine is synthesized by reaction of ethylenediamine with an amino acid ester and reduction of the resulting amide carbonyl. (Brechbiel et al. Inorg. Chem. 25:2772-8 (1986)). Alkylation of the diethylenetriamine with haloacetic acid or ester followed by hydrolysis, if applicable, produces a monosubstituted bifunctional diethylenetriamine pentaacetic acid (DTPA) chelate.
Another method of synthesis of a bifunctional DTPA involves reaction of a DTPA or EDTA carboxylate with a chloroformate ester to form a reactive anhydride (Krejcarek et al., Biochem. Biophys Res. Commun. 77:581 (1977)). The dianhydride of DTPA used as a bifunctional chelate is prepared by dehydration of the parent DTPA (Hnatowich et al., Int. J. Appl. Rad. Isot. 33:327 (1982)). The practice of using an EDTA chelate monosubstituted at the carbon-1 position to better retard the release of metal from chelate in vitro, than the unsubstituted EDTA chelate, has also been reported (Meares et al., Anal. Biochem. 142:68 (1984)).
The prior art has formed metal-protein chelate conjugates by mixing monosubstituted bifunctional EDTA or DTPA chelates or DTPA anhydrides with proteins followed by reaction with the metal to be chelated (Krejcarek et al., Biochem. Biophys. Res. Commun. 77:581, (1977); Brechbiel et al., Inorg. Chem. 25:5783 (1986)). Imaging of tumor target sites in vivo with metal chelate conjugated monoclonal antibodies prepared according to these methods has been reported (Khaw et al., Science 209:295, (1980) Sheinberg et al., Science 215:151, (1982)). Diagnosis of human cancer in vivo using metal chelate conjugated monoclonal antibody has also been reported (Rainsbury et al., Lancet 2:694 (1983)). The use of chimeric antibodies and advantages thereof have been discussed by Morrison, S. L., Hospital Practice 24:64-65, 72-74; 77-80 (1989). The potential efficacy of using a hydrolyzable linking group between a chelate and a protein has also been discussed (Paik et al., J. Nucl, Meal. 30:1693-1701 (1989)).
Disubstituted bifunctional DTPA derivatives have proven useful for labeling of proteins with radioactive metals (Kozak, et al., Cancer Research 49:2639-44 (1989)). The introduction of a second substituent on the carbon backbone of DTPA was seen to retard the loss of metal from the DTPA ligand when linked to antibody and injected into the circulation of animals.
The usefulness of radionuclide materials in cancer therapy is disclosed in the article, Kozak et al., "Radionuclide-conjugated monoclonal antibodies: A Synthesis of Immunology, Inorganic Chemistry and Nuclear Science" Trends in Biotechnology 4:(10):259-264 (1985). This article discusses the use of antibody conjugates to deliver either alpha or beta radiation. The value of alpha radiation for bismuth-212 in radionuclide therapy is further discussed in the two articles; Kozak et al, "Bismuth-212-labeled anti-Tac monoclonal antibody: Alpha-particle-emitting Radionuclides as Modalities for Radioimmunotherapy," Proc. Natl. Acad. Sci. U.S.A. 83:474-478 (1986) and Gansow et al., "Generator-produced Bi-212 Chelated to Chemically Modified Monoclonal Antibody for Use in Radiotherapy," Am. Chem. Soc. Symposium Series 15:215-227 (1984). Ligands, for the secure linkage of bismuth to proteins, have not been available (Macklis et al., Science 240:1024-2 (1988)).
Examples of other uses for chelated metal ions are disclosed in the following articles. Magerstadt et al., "Gd(DOTA): An alternative to Gd(DPTA) as a T.sub.1 /T.sub.2 Relaxation Agent for NMR Imaging or Spectroscopy," Magnetic Resonance in Medicine 3:808-812 (1986), discloses the usefulness of gadolinium as a relaxation agent for NMR imaging. The article, Spirlet et al., Inorgan. Chem. 23:4278-4783(1984), disclosed the usefulness of lanthanide chelates.
All patents and publications referred to herein are hereby incorporated by reference.
However, attempts to employ the tumor localizing properties of metal chelate conjugated monoclonal antibodies for therapeutic purposes have not found common usage because the stability of the radionuclide-linker-antibody conjugate, particularly in vivo over extended time-periods, is of constant concern. This is of special concern when the conjugate is to be used in radioimmunotherapy and contains an alpha- or beta-emitting nuclide. The amount of these highly toxic therapeutic nuclides which can be safely administered is limited by their unwanted dissociation from the antibody-chelate conjugate.
Most therapeutic nuclides are multi-valent heavy metals, and behave physiologically somewhat like iron, a naturally occurring and essential element, noted for its slow absorption into mammalian systems and its virtual negligible excretion, once absorbed. Iron is toxic in high amounts and is never present in circulation in vivo in an uncomplexed form.
The mammalian system goes to great lengths to scavenge free iron and does so by a variety of methods.
One method involves transferrin, an iron transport protein (K.sub.d for Fe.sup.3+ &gt;10.sup.-23). Transferrin will (1) transport iron to bone marrow where synthesis of new red blood cells occurs and (2) deposit iron in the storage protein ferritin for future use. Metals which preferentially bind to the anions of "hard acids", which typically containing oxygen ligands, may be susceptible to the transferrin scavenging system. Such metals may include gallium, indium, yttrium, lutetium, scandium, samarium, and gadolinium.
The degree of susceptibility to transchelation to transferrin will vary among metals, depending on other factors such as ionic radius and the precise nature of the structure of the metal-chelate exposed to the challenge of transferrin.
A distinct metal-scavenging system is based on the protein, metallothionein, which is a 7 kD unit having 21 free cysteine residues. The primary function of this protein is heavy metal detoxification, particularly the scavenging of metals which prefer binding to the anions of "soft acids" which are typified by sulfur containing ligands. Such metals may include copper, zinc, cadmium, silver, mercury and lead.
With these two systems (transferrin and metallothionein), mammals are equipped to regulate the bioavailability of potentially toxic heavy metals, thereby protecting themselves against the effects of undesirable elements. While the administration of antibody-chelate linker-radionuclide conjugates containing trace amounts of metals (carrier-free nuclides) is of little concern toxicologically, the design of metal-bearing antibody conjugates must take into account the mammalian defense/metabolism mechanisms outlined above.
It is evident from the above that there continues to be a need for more effective metal chelate protein conjugates that firmly link metals to proteins to minimize metal release and permit highly selective delivery of metals to targeted sites in vivo.
The purpose of this disclosure is to describe improved agents for radioimmunoscintigraphy and particularly radioimmunotherapy, taking the above observations into account.